| Objective: Previous studies have shown that DNA damage repair is closely related to IKK/NF-κB signaling pathway in breast cancer and gastric cancer cells. NF-κB activity increased in acute myeloid leukemia(AML) and leukemic stem cells,which indicates poor prognosis. Moreover, most chemotherapy drugs generate DNA damage in AML cells. Therefore, this study aimed to investigate whether the IKK / NF-κB signaling pathway can modulate DNA damage repair in AML cells and the related molecular mechanisms. Furthermore, whether inhibition of IKK/NF-κB signaling pathway can enhance the effect of DNA damage drugs on AML cells.Method: The effects of IKKβ inhibitors BMS-345541 on the sensitivity of AML cells to DNR were determined by MTT. DNA damage,cell cycle progression and apoptosis of AML cells were determined by Flow Cytometry(FCM).The amount ofγ-H2 AX,p-ATM,and RAD51 in AML cells were verified by High Content Analysis(HCA) and Western Blot. The effects of BMS345541 on DNA damage repair in sorted human CD34 + leukemia cells was detect by flow cytometry, HCA, and comet assays. Establishment of a stable Rel A knockout KG1 a Rel A(-)cell line by smallhairpin RNA(sh RNA) targeting Rel A. MTT assay,flow cytometry, High Content Analysis(HCA) were applied to detect the difference of DNA damage repair between KG1 a Rel A(-) and KG1 a Vector cells. The effect of Rel A on the DNA damage-repair pathway on KG1 a cells with HR and NHEJ pathway-specific reporter gene. Microarray assay was used to detect differentially expressed long non-coding RNA and m RNA between KG1 a Rel A(-) and KG1 a Vector cells under DNR.Xenograft tumor model of HL-60 cell in nude mice was established to examined whether BMS can also sensitize HL-60 cells to DNR.Results: 1. The results of MTT, FCM, High Content Analysis and comets experiments showed that BMS-345541 efficiently suppressed DNA damage repair,increased apoptosis ratio induced by DNR, reversed the cell cycle arrest in G2/M phases. Its mechanism of action was related to RAD51 aggregation in the DNA breaks, thereby affecting the HR repair; consistent with this, due to the failure to repair DNA damage, DNA damage response signal p-ATM continued to be activated.2. The results of Flow Cytometry, high content and comet assay showed that DNA damage in the combined treatment group was significantly higher than the repair group in CD34 + progenitor / stem cells, which demonstrated that BMS-345541 can effectively block the DNA damage repair of AML progenitor / stem cell.3. Build a stable P65(Rel A) knock down cell line KG1 a Rel A(-).The results of MTT, flow cytometry and HCA experiments showed that Rel A knockdown inhibited DNA damage repair of KG1 a cells, increased KG1 a apoptosis, decreased numbers of cells in the G2/M phases of the cell cycle, and suppressed HR and NHEJ repair pathway in KG1 a cells.4. The results of microarray assay analysis showed that DNA damage repair related genes were significantly reduced, such as PARP1 and XRCC3; moreover, XRCC3 and PARP1 were negatively correlated with Lnc RNA, such as uc010 ixu.3 and uc001 vvr.1.5. BMS can also sensitize HL-60 cells to DNR in a Xenograft tumor model.CONCLUSION:Inhibition of IKK/NF- nuclear factor kappa B signaling pathway can effectively inhibit the DNA damage repair of AML cells, reverse the G2/M phase arrest, and enhance the proliferation inhibition and apoptosis induced by DNR. The possible molecular mechanism is: inhibition of IKK/NF- kappa B signaling pathway activity can prompt DNA damage of AML cells by increasing uc010 ixu.3 and uc001 vvr.1 expression and consequently decrease the expression of PARP1 and XRCC3, which leads to the reduction of DNA damage repair ability of AML cells... |
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